Multifaceted Activities of Seven Nanobodies against Complement C4b

Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined singleparticle cryo-electron microscopy structures in complex with C4b at 3.4-4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody-drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody-drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors

Antibody or Antibody Fragments : Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody-drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody-drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors

Antibody or Antibody Fragments: Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody–drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are clearly still necessary. A major factor limiting the efficacy of antibody-targeted cancer therapies may be the incomplete penetration of the antibody or antibody–drug conjugate into the tumor. Incomplete tumor penetration also affects the outcome of molecular imaging, when using such targeting agents. From the injection site until they arrive inside the tumor, targeting molecules are faced with several barriers that impact intratumoral distribution. The primary means of antibody transport inside tumors is based on diffusion. The diffusive penetration inside the tumor is influenced by both antibody properties, such as size and binding affinity, as well as tumor properties, such as microenvironment, vascularization, and targeted antigen availability. Engineering smaller antibody fragments has shown to improve the rate of tumor uptake and intratumoral distribution. However, it is often accompanied by more rapid clearance from the body and in several cases also by inherent destabilization and reduction of the binding affinity of the antibody. In this perspective, we discuss different cancer targeting approaches based on antibodies or their fragments. We carefully consider how their size and binding properties influence their intratumoral uptake and distribution, and how this may affect cancer imaging and therapy of solid tumors

Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer, although sub-optimal tumor specificity and side effects have hindered its clinical application. We introduced a new strategy named nanobody-targeted PDT in which photosensitizers are delivered to tumor cells by means of nanobodies. As efficacy of targeted PDT can be hampered by heterogeneity of target expression and/or moderate/low target expression levels, we explored the possibility of combined targeting of endothelial and cancer cells in vitro. We developed nanobodies binding to the mouse VEGFR2, which is overexpressed on tumor vasculature, and combined these with nanobodies specific for the cancer cell target EGFR. The nanobodies were conjugated to the photosensitizer IRDye700DX and specificity of the newly developed nanobodies was verified using several endothelial cell lines. The cytotoxicity of these conjugates was assessed in monocultures and in co-cultures with cancer cells, after illumination with an appropriate laser. The results show that the anti-VEGFR2 conjugates are specific and potent PDT agents. Nanobody-targeted PDT on co-culture of endothelial and cancer cells showed improved efficacy, when VEGFR2 and EGFR targeting nanobodies were applied simultaneously. Altogether, dual targeting of endothelial and cancer cells is a promising novel therapeutic strategy for more effective nanobody-targeted PDT

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy

Recent advances in the field of in-cell NMR spectroscopy have made it possible to study proteins in the context of bacterial or mammalian cell extracts or even entire cells. As most mammalian cells are part of a multi-cellular complex, there is a need to develop novel NMR approaches enabling the study of proteins within the complexity of a 3D cellular environment. Here we investigate the use of the hanging drop method to grow spheroids which are homogenous in size and shape as a model system to study solid tumors using solid-state NMR (ssNMR) spectroscopy. We find that these spheroids are stable under magic-angle-spinning conditions and show a clear change in metabolic profile as compared to single cell preparations. Finally, we utilize dynamic nuclear polarization (DNP)-supported ssNMR measurements to show that low concentrations of labelled nanobodies targeting EGFR (7D12) can be detected inside the spheroids. These findings suggest that solid-state NMR can be used to directly examine proteins or other biomolecules in a 3D cellular microenvironment with potential applications in pharmacological research

Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer, although sub-optimal tumor specificity and side effects have hindered its clinical application. We introduced a new strategy named nanobody-targeted PDT in which photosensitizers are delivered to tumor cells by means of nanobodies. As efficacy of targeted PDT can be hampered by heterogeneity of target expression and/or moderate/low target expression levels, we explored the possibility of combined targeting of endothelial and cancer cells in vitro. We developed nanobodies binding to the mouse VEGFR2, which is overexpressed on tumor vasculature, and combined these with nanobodies specific for the cancer cell target EGFR. The nanobodies were conjugated to the photosensitizer IRDye700DX and specificity of the newly developed nanobodies was verified using several endothelial cell lines. The cytotoxicity of these conjugates was assessed in monocultures and in co-cultures with cancer cells, after illumination with an appropriate laser. The results show that the anti-VEGFR2 conjugates are specific and potent PDT agents. Nanobody-targeted PDT on co-culture of endothelial and cancer cells showed improved efficacy, when VEGFR2 and EGFR targeting nanobodies were applied simultaneously. Altogether, dual targeting of endothelial and cancer cells is a promising novel therapeutic strategy for more effective nanobody-targeted PDT

Homogeneous tumor targeting with a single dose of HER2-targeted albumin-binding domain-fused nanobody-drug conjugates results in long-lasting tumor remission in mice

Background: The non-homogenous distribution of antibody-drug conjugates (ADCs) within solid tumors is a major limiting factor for their wide clinical application. Nanobodies have been shown to rapidly penetrate into xenografts, achieving more homogeneous tumor targeting. However, their rapid renal clearance can hamper their application as nanobody drug conjugates (NDCs). Here, we evaluate whether half-life extension via non-covalent interaction with albumin can benefit the efficacy of a HER2-targeted NDC. Methods: HER2-targeted nanobody 11A4 and the irrelevant nanobody R2 were genetically fused to an albumin-binding domain (ABD) at their C-terminus. Binding to both albumin and tumor cells was determined by ELISA-based assays. The internalization potential as well as the in vitro efficacy of NDCs were tested on HER2 expressing cells. Serum half-life of iodinated R2 and R2-ABD was studied in tumor-free mice. The distribution of fluorescently labelled 11A4 and 11A4-ABD was assessed in vitro in 3D spheroids. Subsequently, the in vivo distribution was evaluated by optical molecular imaging and ex vivo by tissue biodistribution and tumor immunohistochemical analysis after intravenous injection of IRDye800-conjugated nanobodies in mice bearing HER2-positive subcutaneous xenografts. Finally, efficacy studies were performed in HER2-positive NCI-N87 xenograft-bearing mice intravenously injected with a single dose (250 nmol/kg) of nanobodies conjugated to auristatin F (AF) either via a maleimide or the organic Pt(II)‑based linker, coined Lx®. Results: 11A4-ABD was able to bind albumin and HER2 and was internalized by HER2 expressing cells, irrespective of albumin presence. Interaction with albumin did not alter its distribution through 3D spheroids. Fusion to ABD resulted in a 14.8-fold increase in the serum half-life, as illustrated with the irrelevant nanobody. Furthermore, ABD fusion prolonged the accumulation of 11A4-ABD in HER2-expressing xenografts without affecting the expected homogenous intratumoral distribution. Next to that, reduced kidney retention of ABD-fused nanobodies was observed. Finally, a single dose administration of either 11A4-ABD-maleimide-AF or 11A4-ABD-Lx-AF led to long-lasting tumor remission in HER2-positive NCI-N87 xenograft-bearing mice. Conclusion: Our results demonstrate that genetic fusion of a nanobody to ABD can significantly extend serum half-life, resulting in prolonged and homogenous tumor accumulation. Most importantly, as supported by the impressive anti-tumor efficacy observed after a single dose administration of 11A4-ABD-AF, our data reveal that monovalent internalizing ABD-fused nanobodies have potential for the development of highly effective NDCs

Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies

PURPOSE: Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. PROCEDURES: The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [111In]DOTA-NB/[177Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. RESULTS: No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid's covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. CONCLUSIONS: This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers